(734c) Design Principles of a Robust Genetic Switch Using Antisense Transcription

Antisense transcription has been extensively recognized as a regulatory mechanism for gene expression across all kingdoms of life. Growing evidence suggests the presence of non-coding cis‑antisense RNAs (AR) that regulate gene expression. Recent studies also indicate the role of transcriptional interference (TI) in regulating expression of neighboring genes due to traffic of RNA polymerases from adjacent promoter regions. While these mechanisms have been described to regulate gene expression in naturally occurring systems, the design principles for antisense transcription have not been explored yet. Here, we combine mathematical modeling and experimental approaches to engineer antisense RNA interaction, RNA polymerase traffic, relative promoter strength and promoter-to-promoter distance in a set of convergent promoters to develop a novel synthetic biology tool. We show that combination of TI and AR adds multiple-levels of regulation which allows such a system to have a highly tunable output in response to stimuli, ranging from a simple-first order response to biologically complex higher-order response such as bistable switch. Using a library of experimental constructs with engineered TI and AR by using face-to-face inducible promoters separated by carefully tailored overlapping DNA sequences we demonstrate tunable control of expression of a set of fluorescent reporter proteins. We show that even subtle changes in the physical arrangement of the convergent promoters can give rise to diverse gene expression patterns, which allows for design of highly-tunable synthetic genetic switches using minimum number of parts.